Recombinant Mouse Transmembrane protein 215 (Tmem215)

What is Transmembrane Protein 215 (TMEM215) and what is its molecular structure?

TMEM215 is an endoplasmic reticulum (ER)-located, 2-pass transmembrane protein. The mouse TMEM215 protein consists of 235 amino acids . Its sequence begins with "MRPDDINPRTGLVVALVSVFLVFGFMFTVSGMKGETLGNI..." and continues through the full 235-residue sequence . TMEM215 is primarily localized to the ER membrane, where it interacts with key regulators of the apoptotic pathway .

Structure characteristics include:

• Total length: 235 amino acids

• Molecular weight: Approximately 25.8 kDa

• Configuration: 2-pass transmembrane protein

• Cellular localization: Endoplasmic reticulum membrane

What are the known biological functions of TMEM215?

TMEM215 serves multiple biological functions depending on the cellular context:

• Endothelial cell survival: TMEM215 protects endothelial cells from apoptosis during vessel pruning and remodeling .

• Regulation of calcium signaling: TMEM215 influences mitochondria-associated ER membrane (MAM) formation and calcium flux from ER to mitochondria .

• Neuronal development: In retinal tissue, TMEM215 marks specific subpopulations of bipolar cells, suggesting a role in neuronal specification or function .

• Response to mechanical stimuli: TMEM215 expression is upregulated by laminar shear stress in endothelial cells via downregulation of EZH2 .

How is TMEM215 expression regulated in different tissues?

TMEM215 expression shows tissue-specific and context-dependent regulation:

• Endothelial cells: Expression is dynamically regulated by blood flow-derived shear stress. Laminar shear stress (LSS) significantly upregulates TMEM215 at both mRNA and protein levels, while oscillatory shear stress (OS) downregulates it compared to LSS .

• Retinal tissue: TMEM215 expression is detected in subpopulations of bipolar cells. In developing retinas, TMEM215 expression is upregulated in the absence of Blimp1 (Prdm1), suggesting regulatory control by this transcription factor .

• Angiogenic vs. quiescent endothelium: TMEM215 expression appears more critical in angiogenic endothelial cells than in quiescent vessels. Studies show that TMEM215 deficiency primarily affects survival in angiogenic endothelial cells, while quiescent endothelial cells in most adult tissues appear less dependent on TMEM215 .

• Vascular beds: Expression is higher in endothelial cells from descending thoracic aorta (with laminar blood flow) compared to those from aortic arch (with turbulent blood flow) .

What is the molecular mechanism by which TMEM215 prevents endothelial cell apoptosis?

TMEM215 prevents endothelial cell apoptosis through a complex molecular pathway involving ER-mitochondria communication and calcium homeostasis:

• BiP interaction: TMEM215 forms a complex with the ER chaperone BiP (binding immunoglobulin protein) and facilitates BiP interaction with the BH3-only proapoptotic protein BIK (BCL-2 interacting killer) .

• Mitochondria-associated ER membrane (MAM) regulation: TMEM215 knockdown leads to:

  • Increased number of MAMs

  • Decreased distance between outer mitochondrial membrane (OMM) and ER membrane

  • Enhanced calcium flux from ER to mitochondria

• Calcium signaling control: TMEM215 regulates mitochondrial calcium influx. Its knockdown increases Ca²⁺ flux from ER to mitochondria, which can be rescued by BIK knockdown .

• Intrinsic apoptosis pathway: TMEM215 knockdown activates the intrinsic apoptosis pathway as evidenced by:

  • Increased cleaved caspase-3 (aCasp3)

  • Increased cleaved caspase-9 (aCasp9)

  • Release of cytochrome C

• BIK dependency: TMEM215 knockdown-induced apoptosis occurs in a BIK-dependent manner and can be abrogated by BCL-2 .

How does TMEM215 contribute to vascular development and remodeling in vivo?

TMEM215 plays critical roles in vascular development and remodeling as demonstrated by conditional knockout studies:

• Retinal vessel remodeling: EC-specific TMEM215 deletion impairs retinal vasculature development in mice, characterized by:

  • Decreased vessel density (ratio of vessel area to vascularized area)

  • Reduced vascular branches leading to reduced complexity in the remodeling zone

  • Significantly decreased number of endothelial cells

  • Increased apoptotic endothelial cells (marked by cleaved caspase-3)

  • More collagen IV⁺/CD31⁻ empty basement membrane sleeves, indicating excessive vessel regression

• Tumor angiogenesis: EC-specific TMEM215 ablation:

  • Inhibits tumor growth

  • Disrupts tumor vasculature

  • Attenuates lung metastasis (consistent with reduced VCAM1 expression)

• Therapeutic potential: Administration of nanoparticles carrying TMEM215 siRNA inhibits:

  • Tumor growth

  • Choroidal neovascularization injury

This suggests TMEM215 as a potential target for anti-angiogenic therapy in pathological conditions .

What experimental challenges exist in studying TMEM215 function across different model systems?

Researchers face several challenges when investigating TMEM215 function:

• Cell-type specificity: TMEM215 knockdown induces apoptosis in endothelial cells but not in other cell types such as 293T cells, HeLa cells, or primary vascular smooth muscle cells, indicating context-dependent function .

• Differential effects in vivo vs. in vitro: While TMEM215 knockdown causes high percentages of endothelial cell apoptosis in vitro, EC-specific knockout in vivo only affects certain vascular beds (retinal vasculature in newborn mice and ovary in adult mice) while other organs appear unaffected .

• Angiogenic vs. quiescent endothelium: TMEM215 deficiency primarily affects angiogenic endothelial cells but not quiescent endothelial cells in most adult tissues, requiring careful experimental design to target specific vascular populations .

• Gene expression effects in quiescent cells: Despite apparent normal survival of quiescent endothelial cells in TMEM215 knockout mice, RNA-seq analysis shows substantial changes in gene expression profiles compared to controls, suggesting complex downstream effects .

• Tissue-specific factors: The signals triggering vessel regression appear to be tissue context-dependent, with blood flow-derived mechanical forces being necessary and sufficient in some contexts but not others .

What experimental approaches are most effective for investigating TMEM215's role in endothelial cell apoptosis?

Based on published research, the following experimental approaches have proven effective:

• Gene knockdown strategies:

  • siRNA or shRNA targeting TMEM215 in human umbilical vein endothelial cells (HUVECs)

  • Combined knockdown of TMEM215 with potential interactors (such as BIK) to assess pathway dependencies

• Apoptosis detection methods:

  • TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay to detect DNA fragmentation

  • Western blotting for apoptotic markers (cleaved caspase-3, cleaved caspase-9, cytochrome C)

  • Immunofluorescence staining for cleaved caspase-3

• Calcium signaling analysis:

  • Measurement of Ca²⁺ flux from ER to mitochondria

  • Inhibition of mitochondrial calcium influx using blockers of:

    • IP₃R (inositol 1,4,5-trisphosphate receptor)

    • MCU (mitochondrial calcium uniporter)

• Mitochondria-ER interaction studies:

  • Transmission electron microscopy (TEM) to visualize MAMs

  • Quantification of MAM number and distance between OMM and ER membrane

• Shear stress experiments:

  • Culture of HUVECs under laminar shear stress using the ibidi apparatus

  • Comparison of gene expression between cells exposed to laminar vs. oscillatory shear stress

How should researchers design in vivo experiments to study TMEM215 function?

The following experimental design considerations are recommended based on successful previous studies:

• Conditional knockout approach:

  • Generate TMEM215-floxed mice

  • Cross with tissue-specific Cre lines (e.g., Cdh5-CreERT2 for endothelial cells)

  • Use tamoxifen induction at appropriate developmental timepoints

• Developmental vascular studies:

  • Administer tamoxifen to P1 (postnatal day 1) pups

  • Analyze retinal vasculature on P7 by whole-mount immunofluorescence

  • Stain for multiple markers:

    • CD31 (endothelial cells)

    • Collagen IV (basement membrane)

    • Cleaved caspase-3 (apoptotic cells)

• Tumor angiogenesis models:

  • Implant tumor cells in conditional knockout mice

  • Assess tumor growth, vascular density, and metastasis

  • Compare with control mice

• Therapeutic targeting:

  • Deliver TMEM215 siRNA using nanoparticles in vivo

  • Evaluate effects in:

    • Tumor models

    • Choroidal neovascularization models

• Comparative analysis across vascular beds:

  • Compare TMEM215 expression between different vascular regions:

    • Descending thoracic aorta (laminar flow)

    • Aortic arch (turbulent flow)

What are the optimal conditions for expressing and purifying recombinant mouse TMEM215?

Based on commercial protocols and research practices, the following approaches are recommended:

• Expression systems:

  • Mammalian expression: HEK-293 cells have been successfully used for mouse TMEM215 expression

  • Cell-free protein synthesis (CFPS) systems offer an alternative approach

• Protein characteristics:

  • Full-length mouse TMEM215: 235 amino acids

  • Molecular weight: ~25.8 kDa

  • Sequence: MRPDDINPRTGLVVALVSVFLVFGFMFTVSGMKGETLGNI... (full sequence available in databases)

• Purification tags:

  • His tag: Enables purification by immobilized metal affinity chromatography

  • Strep tag: Alternative purification approach

• Quality control measures:

  • SDS-PAGE analysis: Visualize protein purity and integrity

  • Western blotting: Confirm identity using anti-tag or anti-TMEM215 antibodies

  • Analytical SEC (HPLC): Assess homogeneity and proper folding

  • Expected purity: >90% for HEK-293 cell expression

• Storage recommendations:

  • Store at -80°C

  • Avoid repeated freeze-thaw cycles

How can TMEM215 research contribute to understanding vascular diseases?

TMEM215 research offers several promising avenues for vascular disease understanding and treatment:

• Pathological angiogenesis:

  • Age-related macular degeneration: TMEM215 inhibition through siRNA nanoparticles reduces choroidal neovascularization

  • Tumor angiogenesis: TMEM215 ablation disrupts tumor vasculature and inhibits tumor growth

• Vessel pruning disorders:

  • TMEM215's role in vessel pruning suggests potential implications for conditions with abnormal vessel regression or persistence

  • EC-specific TMEM215 knockout showed excessive vessel regression in retinal vasculature

• Flow-mediated vascular remodeling:

  • The regulation of TMEM215 by shear stress connects it to flow-dependent vessel remodeling

  • Potential relevance to atherosclerosis, where disturbed flow contributes to disease progression

• Novel anti-angiogenic therapy target:

  • TMEM215 manipulation represents a potential targeted approach for controlling pathological angiogenesis

  • Precise TMEM215 manipulation might effectively target angiogenic ECs while sparing quiescent vasculature

What gene expression changes occur in endothelial cells following TMEM215 manipulation?

Experimental data reveals complex transcriptional changes following TMEM215 manipulation:

• RNA-seq analysis:

  • Quiescent lung ECs from TMEM215 knockout mice show substantial changes in gene expression profile compared with controls

  • TMEM215 expression in retinal ECs correlates negatively with intrinsic apoptosis-related genes

  • TMEM215 expression correlates positively with shear stress response genes Klf2 and Klf4

• VEGF-induced expression:

  • TMEM215 expression is upregulated dose-dependently by VEGF (vascular endothelial growth factor) stimulation in HUVECs

  • This suggests a role in VEGF-mediated angiogenic responses

• Differential effects on pathway components:

  • TMEM215 knockdown activates the intrinsic apoptosis pathway components

  • The extrinsic apoptosis pathway (cleaved caspase-8) or necroptosis markers appear not significantly altered

• VCAM1 regulation:

  • TMEM215 ablation in adult mice reduces VCAM1 expression, potentially explaining reduced metastasis observed in tumor models

What are the emerging techniques for functional characterization of transmembrane proteins like TMEM215?

Several cutting-edge approaches show promise for deeper functional characterization:

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize protein localization within ER microdomains

  • Live-cell imaging to track dynamic protein interactions in real-time

  • Proximity labeling methods (BioID, APEX) to identify interaction partners in native cellular contexts

• Gene editing approaches:

  • CRISPR-Cas9 for precise genome editing to create:

    • Endogenous tagged versions of TMEM215

    • Domain-specific mutations to assess functional importance

    • Conditional alleles for temporal control

• Single-cell analysis:

  • Single-cell RNA-seq to assess heterogeneity in TMEM215 expression across endothelial subpopulations

  • Integration with spatial transcriptomics to map expression patterns in complex tissues

• Structural biology techniques:

  • Cryo-electron microscopy for structural characterization of TMEM215 within membrane complexes

  • Hydrogen-deuterium exchange mass spectrometry to map protein interaction interfaces

• Physiological models:

  • Organ-on-chip technologies to study TMEM215 function under controlled flow conditions

  • 3D vascular organoids to evaluate effects on vessel formation and remodeling in a more physiological context